In the previous funding period we showed that treatment of cultured human keratinocytes with arsenite at submicromolar concentrations induced expression of cyclin D1 and this was accompanied by a shift into the G2 compartment of the cell cycle. Electrophoretic mobility shift assays (EMSA) demonstrated enhanced binding of AP1 and CREB transcription factors to their cognate binding motifs in the cyclin D1 promoter Here we wish to continue the work begun in the previous SCORE funding period by, a) using promoter deletion mutants in luciferase reporter assays to study transcriptional activity and, b) by using chromatin immunoprecipitation (ChIP) as an in vivo assay of transcriptional control of cyclin D1 expression by arsenite. We hypothesize that arsenite is an effector of the MAP kinase stress activated pathway which ultimately activates JNK. To test this we will examine the MAP kinase components of the ERK, JNK and p38 pathways in more detail by using a) chemical inhibitors of components of the MAP kinase pathways and b) dominant negative mutants. Activation of transcription factors resulting from phosphorylation will be demonstrated by western blotting using phosphorylation-specific antibodies. Although our main focus will be on the known components of signaling pathways the possibility exists that arsenite may also bring about phosphorylation of novel elements that may act further upstream. For this reason we also want to examine more global changes in patterns of phosphorylation that occur in response to arsenite using antibody microarrays with a view towards discerning changes in patterns of phosphorylation of proteins that is correlated with the induction of cyclin D1 via transcription factor activation. Finally, we wish to explore the possibility that the inductive effects of arsenite are related to reactive oxygen species as preliminary evidence indicates. If this can be demonstrated this finding will form the basis for future research. PUBLIC HEALTH RELEVANCE: Arsenic is a toxic environmental pollutant and carcinogen. The experiments described are intended to shed light on the molecular mechanism by which arsenic causes cancer in individuals exposed to arsenic over long periods of time. This will ultimately provide targets that may form the basis for new approaches for the prevention of arsenic-caused cancers.